DOI: 10.2478/fv-2018-0011

FOLIA VETERINARIA, 62, 2: 5—12, 2018

ECHINOCHASMUS SWABIENSIS N. SP. (DIGENEA: ECHINOSTOMATIDAE) FROM BLACK KITE (MILVUS MIGRANS MIGRANS) IN DISTRICT,

Suleman, S.1, Khan, M. S.2, Zhu, X. Q.3, Dharejo, A. M.4 Uwalaka, E. C.5, Shah, M.6, Adediran, O. A.7

1Department of Zoology, Hazara University, Mansehra 2Department of Zoology, University of Swabi, Pakistan 3Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences Lanzhou, Gansu Province 730046 China 4Department of Zoology, University of Sindh, Jamshoro Pakistan 5Department of Veterinary Parasitology and Entomology Micheal Okpara University of Agriculture, Umudike Nigeria 6Center for Animal Sciences and Fisheries, University of Swat Pakistan 7Department of Veterinary Parasitology, University of Ibadan Nigeria

[email protected]

ABSTRACT large as the oral sucker. The suckers’ width ratio is 1 : 4.7 to 1 : 5.6. The vitelline follicles are compact and denser at A new species of the genus Echinochasmushas been the lateral sides masking the caeca. This species has been described from the small intestine of the black kite (Mil- added to the record of trematodes circulating among vus m. migrans) collected from Swabi, Khyber Pakh- avian species, especially in the study area. tunkhwa, Pakistan and identified as E. swabiensis n. sp. The new species is different from its congeners in its Key words: Digenea; Echinochasmus swabiensis; body size; it has 22 collar spines which includes two cor- fluke; Milvus migrans; Pakistan; Swabi ner spines on one side, four on the other side and eight marginal plus ventral spines on each side. There areteg- umental-scale like spines interspersed on the anterior INTRODUCTION margin of the ventral sucker with a smaller, terminal oral sucker. The pharynx is nearly twice as large as the The family Echinostomatidae is the largest family of oral sucker, while the ventral sucker is nearly six times as parasitic trematodes, parasitizing birds and mammals in-

5 cluding man [2, 5, 12, 15, 19, 21, 22, 23]. They are charac- then stained in Mayer’s carmalum, dehydrated in gradu- terized by having a head collar with spines and their cer- ated series of ethanol (70—100 %), clarified in xylene and caria encyst in invertebrates like mollusks, insect larvae, mounted permanently in Canada balsam for further study. freshwater snails, crustaceans, and the gills of tadpoles Line drawings were prepared with the aid of a drawing tube and fishes [6, 9, 18, 24, 25]. Echinochasminae is one of ten and photomicrographs were taken by using an Olympus subfamilies of the family Echinostomatidae distinguished DP12 camera. All measurements were given in millime- by having a well-developed head collar with interrupted tres. By using trematodes identification keys and the avail- spines arranged in either a single row or sometimes in able literature [11, 12, 22, 25] the recovered specimens were a double row [4, 21, 25]. placed in the genus Echinochasmus Dietz, 1909. Holotype The genus Echinochasmus Dietz, 1909, is characterized specimens were deposited in the Laboratory of Parasitol- by a well-developed head collar bearing 20—24 or 30— ogy, at Hazara University, Mansehra, Pakistan. 34 spines with the teguments covered by scale like spines, long pre-pharynx, large pharynx, dextral ovary, vitelline field confluent in post-testicular region, a cirrus pouch lying RESULTS anterior to the ventral sucker or overlapping it, and a uterus Table 1. Taxonomic summary which is short with few large eggs when gravid [11, 25].

Species of the genus Echinochasmus Dietz, 1909 are Subfamily Echinochasminae (Odhner, 1910) cosmopolitan in distribution and parasitize birds and Species Echinochasmus swabiensis n. sp mammals [11, 12, 17, 21, 25]. Different authors have re- Host Black kite (Milvus m. migrans, Boddaert) ported different species of the genusEchinochasmus from Site of specimen Small intestine various avian hosts [10, 13, 16, 20]. Different reports also collection exist in Pakistan [1, 3, 7, 8, 23]. This study is aimed at inves- Locality type Yar Hussain, Swabi tigating the presence of flukes in the small intestine of the Number of specimen 4 from a single host, 12 samples examined black kite and describes their morphological characteriza- Frequency 8.33 % tion. This study is the latest addition to the genus Echino- of appearance chasmus recovered from the black kite. Accession number HUPE-1

MATERIALS AND METHODS The morphology ofEchinochasmus swabiensis n. sp. was based on four specimens of these trematodes. The more Twelve apparently healthy, adult Black kites (Milvus clear permanently mounted slides were used for the mor- m. migrans, Boddaert) were trapped alive (July 2012 to phological studies. The head end of these specimens pre- March 2014) from various localities of Swabi (34° 7’ 48" N sented the characteristic features of the genus Echinochas- and 72° 28’ 11" E), Khyber Pakhtunkhwa, Pakistan and mus Dietz, 1909, with a small, triangular mouth opening brought to the laboratory of parasitology at Hazara Uni- and reniform head collar bearing 22 large spines in a single versity, Mansehra, Pakistan. These birds were anesthetized dorsally interrupted row (Figs. 1b and 1d). and necropsied for the presence of trematode parasites. The worms were small, thin, elongated, with a gradu- The entire digestive system with other visceral organs in- ally tapering anterior end and somewhat broadly rounded cluding lungs, liver and heart were removed and placed in posterior end. The total body length was 2.85—2.95 mm separate petri-dishes containing physiological saline. These with a maximum width of 0.525—0.575 mm, at the region organs were examined with the aid of a dissection micro- of acetabulum or its posterior border (Fig. 2a). The anterior scope. Four specimens were found in the contents of the body was 0.95—0.1 mm long while the posterior body was intestine of a single bird. The flukes were removed, placed 1.9—1.95 mm long, so that the anterior body and posterior in saline solution until dead and then fixed in AFA solution body length ratio was 1 : 2. The tegument was covered by (70 % ethanol, 92 ml; formaldehyde, 6 ml; and acetic acid, scale like spines, interspersed only up to the region of the 2 ml) under tolerable pressure of glass slides. They were ventral sucker.

6 The oral sucker was terminal, slightly protruding 0.13—0.134 × 0.13—0.15 mm in size, followed by 0.33 to and much smaller than the pharynx (Fig. 2a), measured 0.5 mm long esophagus which was divided into two intesti- 0.058—0.063 × 0.075—0.08 mm in size; nearly half of the nal caeca at a distance of 0.45—0.59 mm from the anterior size of the pharynx (Fig 2a). The head collar was 0.13— end of the body. The intestinal bifurcation occurred a short 0.20 × 0.29—0.3 mm in size, bearing 2 corner spines on one distance ahead of the ventral sucker and the intestinal caeca side, while 4 on the other side with 8 marginal plus ventral were masked by the lateral fields of the vitellaria (Fig. 2c). spines on each side; so that one side consisted of 12 spines, The ventral sucker was six times larger in length than the while the other had 10 spines (Fig. 2b). The collar spines oral sucker, rounded to oval with well-developed muscular were 0.047—0.06 × 0.018—0.022 mm in size. The corner walls, 0.35—0.38 × 0.35—0.45 mm in size and was situated spines were smaller, 0.028—0.03 × 0.013—0.02 mm in size, in the second quarter of the body at a distance of 0.65 to while the marginal and ventral spines were 0.04—0.075 × 0.83 mm from the anterior end of the body. The width of 0.02—0.023 mm and 0.073—0.075 × 0.022—0.023 mm in the suckers had a ratio of 1 : 4.7 to 1 : 5.6. size, respectively (Fig. 2d). The two testes were with entire margins, post-equato- The pre-pharynx was present but varied in length from rial, median, lying one behind the other, contiguous and 0.05 to 0.063mm, due to the contraction of the neck of the separated by a smaller gap and were sub-equal in size. The flukes (Fig. 2a). The pharynx was nearly twice as large as anterior testis was transversely elongated, somewhat rect- the oral sucker (Fig. 2a), globular to nearly oval, measured angular, 0.11—0.25 × 0.26—0.35 mm in size and was situat-

Fig. 1. Photomicrographs of Echinochasmus swabiensis n. sp. and (c) — entire specimens; (b) and (d) — head collars; (e) — eggs

7 Fig. 2. Diagrams of Echinochasmus swabiensis n. sp. and (c) — whole specimens; (b) and (d) — head collars;(e) — eggs

ed at a distance of 0.6 to 0.65 mm from the posterior border transverse vitelline ducts was situated posterior to the shell of the ventral sucker (Fig. 2a and 2c). The posterior testis gland and anterior to the anterior testis. The vitellaria con- was longer than wide, quadrilateral or nearly triangular sisted of small follicles which were compact and denser at with tapered posterior end and measured 0.325—0.372 × the lateral sides, masking the caeca. The vitelline folds com- 0.3—0.322 mm in size. The distance between the posterior mencing from about the mid portion of the ventral sucker border of the posterior testis and the posterior end of the or its posterior border, ran laterally and met freely behind body was 0.47 to 0.575 mm. The vasa efferentia were incon- the posterior testis. The uterus consisted of a few folds con- spicuous. The cirrus pouch was small, nearly oval, 0.15— taining few, yellowish, large eggs, 0.058—0.078 × 0.046— 0.164 × 0.1—0.13 mm in size and was situated between the 0.06 mm in size (Fig. 1a and 2a). The excretory bladder had intestinal bifurcation and ventral sucker or overlapping the a chamber like appearance and opened into the terminal or anterior border of the ventral sucker. The cirrus was incon- sub-terminal excretory pore (Fig. 2c). spicuous and the genital pore laid between the intestinal bifurcation and the ventral sucker (Fig. 2c). The ovary was rounded to oval in shape, equatorial or DISCUSSION post-equatorial and laid slightly at one side of the body; 0.28 to 0.3 mm away from the posterior border of the ven- The genusEchinochasmus was proposed by Dietz, 1909 tral sucker (Fig. 2a). It was much smaller than the testes and with the type species E. coaxatus, in Europe, Central Asia, measured 0.145—0.15 × 0.1—0.125 mm in size. The shell Siberia [25, 11]. gland was smaller than the ovary, sub-median, situated in Our studied species is larger in size (2.85—2.95 × the close proximity of the ovary. The vitelline reservoir with 0.525—0.575) than E. liliputanus (Looss, 1896) Odh-

8 ner, 1910 (0.5—0.8 × 0.2), E. japonicas Tanabe, 1926 the eggs of E. gorsakii (72—90 × 51—57), E. oligolecithosus (0.6—0.9 × 0.16—0.18), E. microacetabulum Leonov, (73—83 × 54—76), E. milvi (78—84 × 51—57), E. botau- 1958 (0.702—0.936 × 0.28—0.34), E. milvi Yamaguti, 1939 ri (84—97 × 46—65), E. colymbi Shigin in Skrjabin, (0.7—1.2 × 0.27—0.32), E. zubedakhaname Nasir, Di- Bashkirova, 1956 (87 × 60), E. novalichesensis (95.6— etz, 1968 (0.850—1.675 × 0.225—0.555), E. donaldsoni 108 × 58.2—70.7), E. amphiboles (97.2—102.6 × 59.4—67.5), Beaver, 1941 (0.88—1.84 × 0.256—0.32), E. dietzevi Is- E. tobi (102—117 × 60—72), E. antigonus (104—126 × 57— saitschikoff, 1927 (1.13—1.47 × 0.39—0.55), E. bagu- 68) and E. famelicus (110—120 × 70—80). lai Verma, 1935 (1.0—1.75 × 0.25—0.5), E. megavitel- Our studied species’ eggs differ from the eggs of E. be- lus Lal, 1939 (1.05 × 0.45), E. haliasturis Odening, 1962 leocephalus (73—81 × 34—43), E. vindhianae (67 × 40) (1.1—1.9 × 0.8—0.9), E. vindhianae Vasudev, 1973 (1.82— and E. spinosus (74—80 × 40) in width and from the eggs 1.92 × 0.38—0.45), E. spinosus (Odhner, 1910) Skrjabin, of E. muraschkinzewi (81—96 × 43—47), E. cohensi (84— Bashkirova, 1956 (2.02—2.25 × 0.4—0.5), E. coaxatus Di- 89 × 49—52.5) and E. mirus Mendheim, 1940 (92— etz, 1909 (2.13—2.56 × 0.65—0.81) and E. spinulosus Ru- 100 × 4856) in length. dolphi, 1809 (1.09—2.7 × 0.41—0.43). It is however smaller The type species, E. coaxatus Dietz, 1909, is sub-equal in size than E. euryporus Looss, 1896 (3—4 × 0.85), E. no- to our studied species in the size of the pharynx and ovary, valichesensis Tubangui, 1932 (2.32—3.25 × 0.48—0.75), resembling it in the extension of the vitellaria but differs E. botauri Baer, 1923 (3.25 × 0.65), E. amphiboles Kotlan, in having a maximum width in the region of the testes; 1922 (4.16—5.16 × 0.765), E. tobi (6.5—7.5 × 1.1—1.3), 24 larger collar spines (0.0768—0.0816 × 0.019—0.0216) E. fanielicus (Odhner, 1910), Prudhoe, 1944 (8.65 × 1.55) with 3 corner spines and 9 marginal spines on each side; and E. antigonus Gupta, 1955 (11.75—12.92 × 1.2—1.43). cuticle spines scattered up to the posterior level of the ac- Our studied species is also bigger in size than the spe- etabulum; oral sucker two times as large as the oral sucker cies recovered from other avian hosts in Pakistan. These of our studied species; smaller ventral sucker; longer pre- species include E. jamshorensi (0.432 × 0.124), E. mohiuddi- pharynx and esophagus; larger testes lie mid-way between ni (0.780—1.613 × 0.215—0.416), E. passeri (0.725 × 0.285) the ventral sucker and rear end of the body. Moreover, the and E. mazharuddini (0.81—0.88 × 0.39—0.42) described ratio in size of the oral sucker to the acetabulum is about by [3, 7, 8] and [23], respectively. 1:2.6 in E. coaxatus and 1 : 4.7 to 1 : 5.6 in our studied species. Using the number of spines as morphological char- E. mordax (Looss, 1899), Price, 1931 resembles this acteristics, our studied species has a resemblance with new species in the number of spines; size of oral sucker E. cohensi Rao, 1951, E. euryporus, E. gorsakii, 1939, E. mi- and eggs, and extension of the vitellaria; but differs in body croacetabulum, E. milvi, E. mordax (Looss, 1899), Price, shape and arrangement of the collar spines (2 corner and 1931, E. muraschkinzewi Bashkirova, 1941, E. spinosus, 9 marginal spines on each side); having a smaller body with E. spinulosus, E. vindhianae, in having 22 spines; while it a maximum width at the level of the cirrus sac; smaller differs from E. accipiteri Bhutta, Khan, 1975, E. japoni- pharynx and ventral sucker; tegument spines distributed cus, E. botauri, E. beleocephalus, E. bagulai, E. novalichesen- up to the level of the posterior testis but very dense in be- sis, E. megavitellus, E. oligolecithosus Mendheim, 1940, tween the ventral sucker and head; smaller and globular E. tobi, E. militaris Leonov, 1958, E. liliputanus, E. coaxa- testes; cirrus sac oval extending dorsally from the intestinal tus, E. famelicus (Odhner, 1910), Prudhoe 1944, E. am- fork to 2/3 of the length of the acetabulum; the size of the phibolus, E. ruficapensis, E. perfoliatus Ratz, 1908, E. afri- ventral sucker is nearly equal to the size of the pharynx of canum Stiles, 1901, E. jamshorensi, E. mohiuddini which the present studied species. has 24 spines; E. zubedakhaname, E. donaldsoni, E. dietzevi E. bagulai Verma, 1935, differs in having a smaller which has 20 spines; E. passeriwith 26 spines; and E. mazha- body with a maximum width at about the middle of the ruddini with 28 spines. testes; 24 collar spines with 4 corner spines and 8 marginal The eggs of our specimen under study (58—78 × 46—60 spines on each side; the spines on the cuticle extend to the µm or 0.058—0.078 × 0.046—0.06 mm) (Fig. 1e and 2e) are posterior end of the body; the pharynx has the same mean larger in size than those of E. zubedakhaname (42—67 × 32— diameter as the oral sucker; the acetabulum is three times 40) and E. euryporus (67 × 42); while they are smaller than as large as the oral sucker and situated at the commence-

9 ment of the middle third of the body. The oral sucker is laria and position of ventral sucker and gonads, but differs larger, while the pharynx and acetabulum are smaller and in having a smaller body with a maximum width at the re- have 9 to 15 numbers of eggs. gion of the anterior testis; smaller (0.037—0.051 × 0.011— E. vindhianae Asudev, 1973 collected from an Indian 0.015) 22 collar spines (2 corner and 9 marginal on each Tawny eagle, Aquila rapaxvindhiana is similar in having side), tegumental spines covered the body up to the poste- 22 collar spines; maximum width at the level of the acetab- rior margin of the posterior testis; sub-terminal, larger oral ulum; caeca are masked by the vitelline follicles and the vi- sucker; cirrus sac piriform, extending to the posterior bor- telline fields extending anteriorly up to the mid-level of the der of the ventral sucker and smaller ovary. The size ratio acetabulum but differs in having 3 corner spines and 8 mar- of the oral sucker to acetabulum is about 1:2 in E. schwartzi ginal spines on each side of the head collar; the oral sucker and 1 : 4.7 to 1 : 5.6 in our species. and pharynx are sub-equal in diameter; shorter esophagus; E. zubedakhaname Nasir, Diaz, 1968, reported by intestinal fork occurs at a distance of 0.37—0.44 mm from Argumedo, Macedo [14] in 1991 differs in having the anterior end of the body; suckers width a ratio of 1 : 4.2; a smaller body with a maximum width at the level of the and the testes are oval in shape and situated in the middle testes; tegumental spines covered the body up to the pos- of the body. terior margin of the posterior testis; head collar consists E. novalichesensis Tubangui, 1932, collected from the of 20 smaller spines, with 3 corner spines and 7 marginal small intestine of Hyptaenidiatorquata in Novaliches, spines on each side; larger oral sucker, longer esophagus Rizal, Luzon, differs in having somewhat larger body with and smaller pharynx; smaller, equatorial ventral sucker; a maximum width across the anterior testis; larger head suckers width a ratio of 1 : 1.9 to 1 : 2.5; cirrus sac elongated collar bearing 24 spines with 3 corner spines and 9 margin- and situated lateral to the anterior margins of the acetabu- al spines on each side; tegumental spines distributed up to lum; ovary larger, postero-lateral to the acetabulum; vitel- the level of the anterior testis; suckers width a ratio of 1 : 2.7; line fields extra-caecal and exceeds the anterior margin of much smaller pharynx; oval and elongated cirrus pouch; acetabulum. ovary equatorial or pre-equatorial; shell gland median and Our specimen also differs from the species reported larger than ovary. from Pakistan in size and shape of the body; number and E. spinulosus Rudolphi, 1809, syn. Monilifer spinulo- arrangement of collar spines; distribution of vitellaria; size, sus Diets, 1910, differs in having a smaller body; 2 cor- shape and position of gonads and other organs. ner spines and 9 marginal spines on each side of the head E. mohiuddini [7] differs in size and shape of the body; collar; much smaller pharynx (sub-equal to the size of oral number and arrangement of collar spines; size and position sucker); much smaller ventral sucker; suckers with a width of the pharynx; shape, size and position of cirrus sac and ratio of 1 : 2.8 to 1 : 3.2; vitelline fields commencing from shape and size of the testes. E. passeri [8] differs in general the level of the testes and vitelline ducts arise from the an- body shape; number and arrangement of collar spines; size terior end of the vitelline fields; ovary smaller in size and of oral sucker, pharynx and ventral sucker; sucker width situated just before the anterior testis. Differences are also the ratio (1 : 2.06); length of pre-pharynx and esophagus; found in the shape of the testes. shape, size and position of gonads; extension and distribu- E.macrocaudatus Ditrich et al., 1996, differs in hav- tion of vitellaria. E. mazharuddini [23] differs in having ing a smaller body; tegumental spines covered 2/3 of the a smaller body with maximum width at the level of the dorsal surface and ventral surface of the body; smaller col- testes; a smaller head collar bearing 28 spines including lar spines, varies in number, 22 (11+11), 23 (11+12), 24 4 corner spines; longer pre-pharynx; smaller ventral sucker (12+12); larger, sub-terminal oral sucker; a smaller ventral (twice as large as the oral sucker); larger cirrus sac; larger, sucker; smaller pharynx; suckers width a ratio of 1 : 1.68; pear-shaped ovary, overlapped with the acetabulum and ventral sucker situated at about 2/5 of the body length; pre- anterior testis; smaller testes; extension and distribution of pharynx short or absent; cirrus sac elongated and antero- the vitellaira. Moreover, the post-testicular space is 0.122— dorsal to the acetabulum with a large seminal vesicle. 0.139 mm long in E. mazharuddini while 0.47 to 0.575 mm Our studied species is closely related with E. schwartzi long in our species. E. jamshorensi [3] differs in having Price, 1931, in general body shape, extension of the vitel- a smaller body; smaller head collar bearing 24 spines; larger

10 oral sucker; smaller pharynx; much smaller ventral sucker; matidae) from Paddy Bird Ardeolagrayii (Ardeidae) of Hy- smaller cirrus sac; smaller gonads and longer post-testicu- derabad, Sindh, Pakistan. Proc. Parasitol., 39, 285—288. lar space. 8. Dharejo, A. M., Birmani, N. A., Khan, M. M., 2010: Echi- nochsmus passeri, new species (Digenea: Echinostomati- dae) from gallbladder of House Sparrow, Passer domesticus CONCLUSIONS (Aves: Passeridae) of Hyderabad, Sindh, Pakistan. Proc. Parasitol., 50, 139—145. The variations in our studied specimens from the pre- 9. Dimitrov, V., Kanev, I., Bezprozvanich, V., Radev, V., 1998: viously described species of the genus Echinochasmus, Argentophilic structures of the miracidium of Echinochas- strengthen the statement that the specimens discovered mus perfoliatus (Trematoda: Echinosmatidae). Parasite, 5, in our investigation are new to science. This also calls for 185—188. more research into this particular family especially in the 10. Faltynková, A., Gibson, D. I., Kostadinova, A., 2008: A revi- area of its systematics and epidemiology. The new species is sion of Petasiger Dietz, 1909 (Digenea: Echinostomatidae) named as Echinochasmus swabiensis. and a key to its species. Syst. Parasitol., 71, 1—40. Etymology: Name of new species refers to the locality 11. Gibson, D. I., Jones, A., Bray, R. A., 2005: Keys to the Trema- of the host. toda, Vol. 2., CABI Publishing and the Natural History Mu- “Nomenclatural acts”: This work and the nomenclatur- seum, Wallingford and London, UK, 745 pp. al acts it contains have been registered in the Zoo Bank. The 12. Kostadinova, A., Jones, A., 2005: Superfamily Echinosto- Zoo Bank Life Science Identifier (LSID) for this publication matoidea. In Jones, A., Bray, R. A., Gibson, D. I.: Keys to the is:LSIDurn:lsid:zoobank.org:pub:7F675C67-1A8B-4CC3- Trematoda, Vol. 2., CABI Publishing and the Natural History B143-653CD3EDD107. Museum, Wallingford and London, UK, 5—8. 13. Kudlai, O., Kostadinova., A, Pulis, E. E., Tkach, V. V., 2015: A new species of Drepanocephalus Dietz, 1909 (Digenea: REFERENCES Echinostomatidae) from the Double-crested Cormo- rant Phalacrocoraxauritus (Lesson) (Aves: Phalacrocoraci- 1. Bhutta, M. S., Khan, D., 1975: Digenetic trematodes of verte- dae) in North America. Syst. Parasitol., 90, 221—230. brates from Pakistan. Bull. Dept. Zool. Univ. Punjab, 8, 1—175. 14. Argumedo, R. L., Macedo, L. A., 1991: Trematodos de aves 2. Chai, J. Y., Lee, S. H., 2002: Food borne intestinal trematode IV. Estudio de Echinochasmus zubeddakhanamae (Trematoda: infections in the republic of Korea. Parasitol. Int., 51, 129—154. Echinostomatidae) recuperados experimentalmente. An- 3. Channa, M. A., Khan, M. M., Shaikh, A. A., Dharejo, A. M., nales Inst. Biol. Univ. Nac. Auton. Mex. Ser. Zool., 62, 11—14. 2009: Echinochasmu sjamshorensi, new species (Tremato- 15. Leles, D., Cascardo, P., dos Santos Freire, A., Maldonado, da: Echinostomatidae) from Pond Heron, Ardeolagrayii A., Sianto, L., Araujo, A., 2014: Insights about echinostomia- (Aves: Ardeidae) of Jamshoro, Sindh, Pakistan. Proc. Parasi- sis by paleomolecular diagnosis. Parasitol. Int., 63, 646—649. tol., 48, 151—158. 16. Overstreet, R. M., Cook, J. O., Heard, R. W., 2009: Trema- 4. Choi, S., Lee, D., Park, H., Oh, M., Jeon, H. K., Lee, Y., Eom, toda (platyhelminthes) of the Gulf of Mexico. In Felder, D. L., K. S., 2014: Three Echinostome species from wild birds in the Camp, D. K.: Gulf of Mexico-Origins, Waters and Biota Bio- Republic of Korea. Korean J. Parasitol., 52, 513—520. diversity. Texas A and M University Press, College station, 5. Choi, M. H., Kim, S. H., Chung, J. H., Jang, H. Y., Joon, H. E., Texas, 419—486. Chung, B. S., 2006: Morphological observations of Echino- 17. Platt, T. R., 2006: First report of Echinochasmus sp. from the chasmus japonicas cercariae and the in vitro maintenance of snapping turtle (Chelydraserpentina L.) from Reelfoot Lake, its life cycle from cercariae to adults. J. Parasitol., 92, 236—241. Tennessee, USA. Comp. Parasitol., 73, 161—164. 6. Detwiler, J. T., Zajac, A. M., Minchella, D. J., Belden, L. K., 18. Scholz, T., Ditrich, O., Vargas-Vázquez, J., 1996: Echino- 2012: Revealing cryptic parasite diversity in a definitive host: chasmus leopoldinae n. sp. (Trematoda: Echinostomatidae) echinostomes in muskrats. J. Parasitol., 98, 1148—1155. and data on its life-cycle. Syst. Parasitol., 33, 157—165. 7. Dharejo, A. M., Bilqees, F. M., Khan, M. M., 2007: Echino- 19. Seo, B. S., Lee, S. H., Chai, J. Y., Hong, S. J., 1985: Studies on chasmus mohiuddini, new species (Trematoda: Echinosto- intestinal trematodes in Korea XX. Four cases of natural hu-

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